Disease: Trypansoma brucei - sleepy sickness Enzyme:Protein Phosphatase 2B Target: T. brucei TREU927 protein phosphatase 2B NCBI Gene #: 71747666 Protein ID#:XP_822888
Background/Disease Information:
Sleeping sickness, also known as African trypanosomiasis, is a vector-borne disease caused by the protozoa of the Trypanosoma brucei species. The tsetse fly is the insect that is responsible for transmitting the disease, and can only be found in the 36 sub-Saharan Africa countries. There are two forms in which the Trypanosoma brucei species can cause sleeping sickness in humans: Trypanosoma brucei gambiense andTrypanosoma brucei rhodesiense. Trypanosoma brucei gambiense formaccounts for 98% of the reported sleeping sickness and located in West Africa while Trypanosoma brucei rhodesiense accounts for 2% of the reported sleeping sickness and is located in East Africa (1). The Eastern African form of sleeping sickness has shown to be more progressive than the Western African sleeping sickness (1, 2). Sleeping sickness is a potentially fatal disease that has become a public health care concern. It consists of two stages; the first has symptoms that include fever, headache and joint pain. The second stage is the neurological phase in which the parasite crosses the blood brain barrier and infects the central nervous system. This stage causes the disruption of the sleep cycle where sleep occurs during the daytime and wakefulness during nighttime (3). Other symptoms for this stage include tremor, confusion, hemiparesis, and general muscle weakness. Without treatment the disease is fatal (3, 4). Treatment in place today for this disease is pentamidine for stage one and melarsoprol, eflornithine, nifurtimox, and nifurtimox-eflornithine for stage two sleeping sickness (3). These treatments options are toxic however. In fact melarsoprol is the only drug that has been proven successful against both types of central nervous system disease, but is so toxic that it kills 5% of patients who receive it (5).Therefore there is a pressing need for a drug that will successfully treat both stages of this disease.
pl: 6.0436; Amino Acid Length: 431; MW: 48129 (Da); Number of Paralogs: 1 Essentiality: Essential, used for cytokinesis Gene/Ortholog: Tb09.160.0480 (OG4_30044);Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford Metabolic Pathways: gene is not mapped to metabolic pathways Not complex
Reaction catalyzed by protein phosphatase 2B (from Brenda):
Crystal Structure Image of Protein (PyMol):
Fig. 1. ribbon structure of Protein phosphatase 2B also known as Calceneurin
Molecular Weight in kiloDaltons using the Expasy ProtParamwebsite: 48128.6 Da Molar Extinction coefficient at 280 nm wavelength: 45350 - assuming all pairs of Cys residues form cystines 44350 - assuming all Cys residues are reduced
Primer Design Results: Forward Primer (33 base pairs) TACTTCCAATCCATGTCTTCTGGCGCGTCCCAT (order) Melting Temperature 0 mM Mg 66.8 degC 1.5 mM Mg 73.6 degC 2 mM Mg 74.1 degC 4 mM Mg 75.0 degC 6 mM Mg 75.4 degC Reverse Primer (38 base pairs) GCGCAATGCACCACATTGTTACCCAGTAAAGGTGGATA Reverse complement TATCCACCTTTACTGGGTAACAATGTGGTGCATTGCGC (order) Melting Temperature 0 mM Mg 66.4 degC 1.5 mM Mg 73.7 degC 2 mM Mg 74.1 degC 4 mM Mg 74.9 degC 6 mM Mg 75.3 degC
Keely: Forward Primer(30bp)
TACTTCCAATCCATGTCTTCTGGCGCGTCC
Reverse Primer:
CACCACATTGTTACCTAACAGTAAAGGTGGATA
Reverse Complement(33bp)
TATCCACCTTTACTGTTAGGTAACAATGTGGTG
DNA Sequence from DNA Works with a TAA added to the end:
Enzyme: Protein Phosphatase 2B
Target: T. brucei TREU927 protein phosphatase 2B
NCBI Gene #: 71747666
Protein ID#:XP_822888
Background/Disease Information:
Sleeping sickness, also known as African trypanosomiasis, is a vector-borne disease caused by the protozoa of the Trypanosoma brucei species. The tsetse fly is the insect that is responsible for transmitting the disease, and can only be found in the 36 sub-Saharan Africa countries. There are two forms in which the Trypanosoma brucei species can cause sleeping sickness in humans: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Trypanosoma brucei gambiense formaccounts for 98% of the reported sleeping sickness and located in West Africa while Trypanosoma brucei rhodesiense accounts for 2% of the reported sleeping sickness and is located in East Africa (1). The Eastern African form of sleeping sickness has shown to be more progressive than the Western African sleeping sickness (1, 2).
Sleeping sickness is a potentially fatal disease that has become a public health care concern. It consists of two stages; the first has symptoms that include fever, headache and joint pain. The second stage is the neurological phase in which the parasite crosses the blood brain barrier and infects the central nervous system. This stage causes the disruption of the sleep cycle where sleep occurs during the daytime and wakefulness during nighttime (3). Other symptoms for this stage include tremor, confusion, hemiparesis, and general muscle weakness. Without treatment the disease is fatal (3, 4). Treatment in place today for this disease is pentamidine for stage one and melarsoprol, eflornithine, nifurtimox, and nifurtimox-eflornithine for stage two sleeping sickness (3). These treatments options are toxic however. In fact melarsoprol is the only drug that has been proven successful against both types of central nervous system disease, but is so toxic that it kills 5% of patients who receive it (5).Therefore there is a pressing need for a drug that will successfully treat both stages of this disease.
pl: 6.0436; Amino Acid Length: 431; MW: 48129 (Da); Number of Paralogs: 1
Essentiality: Essential, used for cytokinesis
Gene/Ortholog: Tb09.160.0480 (OG4_30044); Phenotype: significant loss of fitness in bloodstream forms (3 days); Source study: alsford
Metabolic Pathways: gene is not mapped to metabolic pathways
Not complex
EC#: 3.1.3.16
Drugability: 0.8 (yes)
Link to Brenda:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
BLAST:
Query Coverage: 100%
Max % Identities: 100%
Current Inhibitors: no drug identifiers are listed
Expression Information (has it been expressed in bacterial cells): no expression information available
Purification Method:
Assay:
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/calcineurin.Par.0001.File.tmp/calcineurin.pdf
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
GC % Content for gene: 47.4%
Reaction catalyzed by protein phosphatase 2B (from Brenda):
Crystal Structure Image of Protein (PyMol):
Molecular Weight in kiloDaltons using the Expasy ProtParamwebsite: 48128.6 Da
Molar Extinction coefficient at 280 nm wavelength:
45350 - assuming all pairs of Cys residues form cystines
44350 - assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
Protein Sequence:
MSSGASHHELTRGRDGLKEREYVWKKSHADESPGSNPQILPTLVLPHHLVFDNDGAPLAD
NIKVHFGRGWRLHVEDALNIVHRCALIMKEEPNVVRLKGSAVVCGDLHGQFHDLLTLLEV
NGHPSVQQYVFLGDYVDRGDFSAEIVLLCMSFKLLYPRSFILLRGNHESRQLTSCFNFKQ
EIESKYSSMVYEEIMAAFDCFPLSCVVNDRFFCVHGGLSPLLTYLGEIDTVNRFRETPST
GPMCDLLWSDPMFGDDTDCATPSEELFVFNTKRGCSYNYSYEAVCRFLEANNLCTVIRGH
ETQPGGYKLYRHTPKGVPAVVCVFSASNYCGTYGNMAAVVAIDGDVMNIRQYMATSHDSC
TPNHFNAISRMQPLAIHEAVEKWCVASHGGSSGDAKAEKVEVEKNLSEDDSSVVLREKLG
DMICAMHHIVT
Sequence Length: 431 aa
Coding Sequence:**
GAATATGTTTGGAAAAAATCTCATGCAGATGAATCTCCCGGCAGTAATCCTCAGATACTT
CCGACTCTCGTGTTGCCGCACCATTTGGTATTTGATAATGATGGCGCCCCGCTTGCAGAT
AATATTAAAGTTCATTTTGGGAGAGGATGGCGACTTCATGTAGAGGATGCATTGAACATT
GTCCACCGCTGTGCATTAATCATGAAAGAGGAGCCAAATGTTGTACGATTAAAGGGATCT
GCTGTCGTGTGTGGGGATCTTCACGGGCAGTTTCATGACTTACTGACATTGTTAGAAGTT
AATGGTCATCCCAGCGTGCAGCAATATGTTTTTCTTGGAGACTACGTGGACCGCGGGGAT
TTTAGTGCAGAAATTGTACTCCTCTGCATGTCGTTTAAGCTACTGTACCCTCGTTCCTTC
ATACTACTACGTGGCAATCACGAATCACGACAACTAACGTCGTGTTTCAACTTCAAGCAG
GAGATTGAGAGCAAGTATTCGTCAATGGTGTACGAAGAGATTATGGCAGCGTTTGACTGC
TTTCCACTCTCATGTGTTGTAAATGATAGGTTTTTCTGTGTGCACGGCGGTTTATCGCCA
TTGTTAACTTATCTTGGAGAAATTGACACTGTTAACCGTTTCAGGGAAACACCGTCGACG
GGTCCCATGTGCGATTTGCTCTGGTCTGATCCCATGTTTGGAGACGACACCGACTGTGCA
ACTCCCTCTGAAGAGTTGTTTGTTTTCAATACCAAACGTGGTTGCTCATACAACTACAGT
TATGAGGCAGTTTGTCGGTTTCTGGAGGCGAATAATCTTTGCACAGTCATACGTGGCCAC
GAGACGCAGCCGGGAGGTTACAAACTGTATCGTCACACCCCGAAGGGAGTTCCAGCTGTG
GTTTGTGTTTTTTCCGCATCGAATTACTGTGGAACGTATGGAAATATGGCTGCCGTGGTA
GCAATTGATGGGGACGTGATGAATATTCGTCAATATATGGCGACCTCCCATGACTCCTGC
ACTCCAAATCATTTTAATGCAATTAGTCGTATGCAACCATTGGCAATACACGAGGCAGTT
GAAAAATGGTGTGTGGCTTCACACGGCGGAAGTAGTGGTGATGCAAAGGCAGAAAAGGTG
GAAGTAGAAAAAAATCTTTCGGAGGATGATTCTTCTGTGGTACTGCGGGAGAAGTTGGGG
GATATGATTTGTGCGATGCATCATATAGTTACTTGA
Sequence Length: 1296 bp
atgagcctggatgcgctgaccaccctgccgattaaaaaacataccgcgctgctgaaccgc tttccggaaacccgctttgtgacccagctggcgaaaaaacgcgcgagctggattgtgttt ggccattatctgaccccggcgcagtttgaagatatggatttttttaccaaccgctttaac gcgattctggatatgtggaaagtgggccgctatgaagtggcgctgatggatggcgaactg accagcgaacatgaaaccattctgaaagcgctggaactggattatgcgcgcattcaggat gtgccggatctgaccaaaccgggcctgattgtgctggatatggatagcaccgcgattcag attgaatgcattgatgaaattgcgaaactggcgggcgtgggcgaagaagtggcggaagtg accgaacgcgcgatgcagggcgaactggattttgaacagagcctgcgcctgcgcgtgagc aaactgaaagatgcgccggaacagattctgagccaggtgcgcgaaaccctgccgctgatg ccggaactgccggaactggtggcgaccctgcatgcgtttggctggaaagtggcgattgcg agcggcggctttacctattttagcgattatctgaaagaacagctgagcctggattatgcg cagagcaacaccctggaaattgtgagcggcaaactgaccggccaggtgctgggcgaagtg gtgagcgcgcagaccaaagcggatattctgctgaccctggcgcagcagtatgatgtggaa attcataacaccgtggcggtgggcgatggcgcgaacgatctggtgatgatggcggcggcg ggcctgggcgtggcgtatcatgcgaaaccgaaagtggaagcgaaagcgcagaccgcggtg cgctttgcgggcctgggcggcgtggtgtgcattctgagcgcggcgctggtggcgcagcag aaactgagctggaaaagcaaagaaggccatcatcatcatcatcat
Link to DNA works output file:
https://drive.google.com/?tab=wo&authuser=0#folders/0B4O2KqKh2q_-MlZNOWY4YWJJV0E
Primer Design Results:
Forward Primer (33 base pairs)
TACTTCCAATCCATGTCTTCTGGCGCGTCCCAT (order)
Melting Temperature
0 mM Mg 66.8 degC
1.5 mM Mg 73.6 degC
2 mM Mg 74.1 degC
4 mM Mg 75.0 degC
6 mM Mg 75.4 degC
Reverse Primer (38 base pairs)
GCGCAATGCACCACATTGTTACCCAGTAAAGGTGGATA
Reverse complement
TATCCACCTTTACTGGGTAACAATGTGGTGCATTGCGC (order)
Melting Temperature
0 mM Mg 66.4 degC
1.5 mM Mg 73.7 degC
2 mM Mg 74.1 degC
4 mM Mg 74.9 degC
6 mM Mg 75.3 degC
Keely:
Forward Primer(30bp)
TACTTCCAATCCATGTCTTCTGGCGCGTCC
Reverse Primer:
CACCACATTGTTACCTAACAGTAAAGGTGGATA
Reverse Complement(33bp)
TATCCACCTTTACTGTTAGGTAACAATGTGGTG
DNA Sequence from DNA Works with a TAA added to the end:
ATGTCTTCTGGCGCGTCCCATCATGAGCTGACCCGTGGTCGTGATGGTCTCAAAGAACGT GAATACGTTTGGAAAAAATCTCACGCGGACGAGTCCCCGGGTTCTAACCCGCAAATCCTG CCGACCCTGGTTCTGCCGCACCACCTCGTGTTCGACAACGACGGTGCGCCACTGGCGGAC AACATCAAAGTTCACTTCGGTCGCGGTTGGCGCCTCCACGTTGAGGATGCGCTGAATATC GTTCACCGTTGCGCGCTGATCATGAAGGAGGAACCGAATGTTGTACGCCTGAAGGGCTCT GCAGTAGTCTGCGGTGATCTCCACGGCCAGTTCCACGACCTCCTGACCCTGCTGGAAGTT AATGGTCACCCTAGCGTTCAACAGTACGTTTTCCTGGGTGACTACGTTGACCGTGGTGAC TTCTCTGCTGAAATCGTTCTGCTGTGCATGTCTTTCAAACTGCTGTACCCGCGCTCTTTC ATCCTGCTGCGTGGTAACCACGAGTCTCGCCAGCTGACGTCTTGCTTCAACTTCAAACAG GAAATCGAATCTAAATACTCTTCTATGGTTTACGAAGAAATCATGGCGGCGTTCGACTGC TTCCCGCTGTCTTGCGTTGTTAACGACCGTTTCTTTTGTGTTCATGGTGGTCTCTCTCCG CTGCTGACCTACCTCGGTGAGATCGATACTGTTAACCGTTTTCGTGAAACCCCGTCTACT GGTCCGATGTGTGACCTGCTCTGGTCTGATCCAATGTTCGGTGATGACACCGACTGTGCG ACGCCGAGCGAAGAACTGTTCGTTTTTAACACTAAACGTGGTTGCTCTTACAACTACAGC TACGAAGCCGTTTGCCGTTTCCTCGAAGCGAACAATCTGTGCACGGTTATTCGTGGTCAT GAGACCCAGCCGGGTGGTTATAAACTGTACCGCCATACCCCAAAGGGTGTTCCGGCGGTT GTATGTGTTTTTAGCGCGAGCAATTATTGCGGTACCTACGGTAACATGGCCGCAGTTGTA GCGATCGATGGTGACGTTATGAACATCCGTCAGTACATGGCGACCTCTCACGACTCTTGC ACCCCTAACCACTTCAATGCGATCTCTCGTATGCAGCCTCTGGCGATCCATGAGGCGGTA GAAAAGTGGTGTGTTGCGTCTCACGGTGGTTCTTCCGGTGACGCGAAAGCGGAAAAAGTT GAGGTTGAAAAAAACCTGTCTGAAGATGACAGCTCTGTTGTTCTGCGTGAAAAACTGGGC GATATGATCTGCGCAATGCACCACATTGTTACCTAA
References: